TBX2 affects proliferation, apoptosis and cholesterol generation by regulating mitochondrial function and autophagy in bovine cumulus cell

Abstract Background T‐box transcription factor 2 (TBX2) is a member of T‐box gene family whose members are highly conserved in evolution and encoding genes and are involved in the regulation of developmental processes. The encoding genes play an important role in growth and development. Although TBX2 has been widely studied in cancer cell growth and development, its biological functions in bovine cumulus cells remain unclear. Objectives This study aimed to investigate the regulatory effects of TBX2 in bovine cumulus cells. Methods TBX2 gene was knockdown with siRNA to clarify the function in cellular physiological processes. Cell proliferation and cycle changes were determined by xCELLigence cell function analyzer and flow cytometry. Mitochondrial membrane potential and autophagy were detected by fluorescent dye staining and immunofluorescence techniques. Western blot and quantitative real‐time reverse transcription polymerase chain reaction (qRT‐PCR) were used to detect the expression changes of proliferation and autophagy‐related proteins. Aadenosine triphosphate (ATP) production, glucose metabolism, and cholesterol synthesis of cumulus cells were measured by optical density and chemiluminescence analysis. Results After inhibition of TBX2, the cell cycle was disrupted. The levels of apoptosis, ratio of light chain 3 beta II/I, and reactive oxygen species were increased. The proliferation, expansion ability, ATP production, and the amount of cholesterol secreted by cumulus cells were significantly decreased. Conclusions TBX2 plays important roles in regulating the cells’ proliferation, expansion, apoptosis, and autophagy; maintaining the mitochondrial function and cholesterol generation of bovine cumulus cells.

Conclusions: TBX2 plays important roles in regulating the cells' proliferation, expansion, apoptosis, and autophagy; maintaining the mitochondrial function and cholesterol generation of bovine cumulus cells.

K E Y W O R D S
autophagy, cumulus cells, physiological function, reactive oxygen species, T-box transcription factor 2 INTRODUCTION Improving the quality of oocytes is important for animal husbandry and human fertility (Xu et al., 2018) because the quality of oocytes is the primary factor affecting the fertilisation and breeding of healthy offspring (Keefe et al., 2015). As a subgroup of granular cells, cumulus cells play important role in the nutrition and maturation of oocytes (Dumesic et al., 2015). Oocytes are coupled to surrounding cumulus cells through interstitial junctions (Petro et al., 2012), and this highly specific membrane junction forms the regulatory system that mediates the intercellular transfer of metabolites and regulatory molecules (Lin et al., 2016). Under normal physiological conditions, oocytes generate many factors that regulate cumulus cells, and cumulus cells provide nutrients for oocyte growth through an intracellular exchange; these processes are interdependent but closely related (Gilchrist et al., 2008). In addition, oocytes, under pathological conditions, protect themselves against oxidative stress through antioxidant-scavenging enzymatic (e.g., involving catalase and glutathione peroxidase) and nonenzymatic (e.g., involving ascorbic acid and reduced glutathione) networks provided by the surrounding cumulus cells (Cetica et al., 1999(Cetica et al., , 2001Shaeib et al., 2016).
As intracellular energy factories, mitochondria are the main producers of intracellular reactive oxygen species (ROS) (Oyewole & Birch-Machin, 2015), which are involved in processes such as cell differentiation, cell signal transmission, cell apoptosis, and the regulation of cell growth and the cell cycle. When excessive production of ROS occurs, the accumulation of oxidants exceeds the cell's ability to clear them, and the oxidation system and antioxidant system become unbalanced. A sharp increase in ROS will lead to oxidative stress, causing cell damage and apoptosis . In the process of cell apoptosis, the ratio of BAX/BCL2 (Kulsoom et al., 2018;Lossi et al., 2018) directly determines the degree of opening of various channels in the mitochondrial outer membrane, and BAX and BCL2 represent a regulatory hub of cell apoptosis, while Caspase 3 usually co-regulates apoptosis with BAX and BCL2 (Zhao et al., 2018). In addition, preferential autophagy of damaged or excess organelles such as peroxisomes, the endoplasmic reticulum and mitochondria can occur in response to ROS (Scherz-Shouval & Elazar, 2007). ROS-mediated autophagy and apoptosis of cumulus cells will affect their secretory functions, thus potentially affecting the development and quality of oocytes (Adriaenssens et al., 2010;Huang & Wells, 2010).
T-box gene family is a phylogenetically conserved family of genes that share a common DNA-binding domain (Chapman et al., 1996) and is important in the regulation of body development. Research has shown that T-box transcription factor 2 (TBX2) functions as a transcriptional repressor during germ layer formation, and this activity is mediated in part through repression of target genes stimulated in the mesendoderm by transactivating T-box proteins (Teegala et al., 2018).
In addition, TBX2, as a transcription factor, is involved in embryonic development and cell cycle regulation and inhibits the cycle regulation factors p21 and p14 to make cells resist senescence (Abrahams et al., 2010;Jacobs et al., 2000;Peres et al., 2010). Recent research also showed that after the TBX2 gene was knocked down, cell proliferation and invasion were significantly decreased; after TBX2 was overexpressed, cyclin E and the phosphorylated extracellular signal-regulated kinase levels were upregulated (Liu et al., 2019).
Although TBX2 has been extensively studied in cancer cells (Crawford et al., 2019), its biological functions in bovine cumulus cells remain unclear. This study investigated the effects of TBX2 on ROS levels, mitochondrial function, and cell proliferation in cumulus cells by inhibiting the expression of TBX2. The results will provide a new basis for understanding the biological roles of TBX2 as well as cumulus cells.

MATERIALS AND METHODS
The chemicals and reagents that we used in the experiment were bought from Sigma-Aldrich except expressly stated elsewise in the article.

siRNA treatment
A total of 1 × 10 5 cells were seeded into six-well plates (Nest Biotechnology) containing cell culture medium for 24 h. Then, TBX2-specific siRNA (si-TBX2) and negative control scrambled siRNA (si-NC; no effects on known mammalian gene expression) (GenePharma Co. Ltd.) were administered with RiboFECTCP (Guangzhou RiboBio Co. Ltd.) reagent into cells at a confluence of approximately 70% according to the manufacturers' instructions and our previous study . Then, the cells were incubated for 48 h at 38.5 • C in a 5% CO 2 incubator without changing the culture medium. The specific sequences of the siRNAs are shown in Supplementary Table S1.

Cell cycle assay
In brief, 1 × 10 5 cells were seeded in six-well culture plates. After siRNA treatment for 48 h, the cell cycle distribution was determined using a Cell Cycle and Apoptosis Analysis Kit (Beyotime) and a flow cytometer (Beckman Coulter) according to the manufacturers' instructions.
The data were processed by using MODFIT software (Verity Software House).

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted using Tripure Isolation Reagent (Roche). cDNA was synthesised from the extracted RNA with a reverse transcription kit (Tiangen) according to the instructions. Gene expression was quantified with a Mastercycler ep realplex system (Eppendorf) and the 2 −ΔΔCt method with β-actin as the reference gene using the following protocol: 95 • C for 3 min; 40 cycles at 95 • C for 30 s, 60 • C for 30 s and 72 • C for 30 s. All primers used are listed in Supplementary Table   S1.

Cell proliferation assay
The cell proliferation was assayed using xCELLigence system (Roche Applied Science and ACEA Biosciences) as described previously with some modifications (Bird & Kirstein, 2009;Urcan et al., 2010). In brief, 50 μl of cell culture media with si-TBX2 or si-NC at room temperature was added into each well of E-plate 16. Then, the E-plate 16 was connected to the system and checked in the cell culture incubator for proper electrical contacts, and the background impedance was measured. Meanwhile, the cells were resuspended in a cell culture medium with siRNAs. Hundred microlitres of each cell suspension containing 5 × 10 3 cells was added to the 50 μl medium containing si-TBX2 or si-NC on E-plate 16 in order to determine the optimum cell concentration.
After 30 min incubation at room temperature, E-plate 16 was placed into the cell culture incubator. Finally, cell proliferation was monitored every 30 min for a period of up to 40 h via the incorporated sensor electrode arrays of the E-Plate 16. The electrical impedance was measured by the Real Time CelI AnaIysis (RTCA)-integrated software of the xCELLigence system as a dimensionless parameter termed cell index.

Apoptosis detection
The apoptosis of cumulus cells was detected according to the instruc- The cell samples were then analysed using a flow cytometer (Beckman Coulter).

Mitochondrial membrane potential (MMP) measurement
Briefly, the high-transparency cell culture slides (Nest Biotechnology; Cat. No. #801009) were placed into the six-well plate before the cells were cultured. After siRNA treatment, the culture medium was removed, and the cell slides were washed twice with PBS.
The red and green fluorescence intensities were observed with a fluorescence microscope (Olympus). The red and green fluorescence and the ratio between the average optical densities in each sample were analysed by Image-Pro Plus software (Media Cybernetics) according to its manual (https://www.mediacy.com/images/ipplus/IPPStartUp.pdf).
The relative fluorescence intensity level was measured as the average fluorescence intensity of individual cells (JC-1 red /JC-1 green ) from three independent experiments with three randomly selected observation images.

Determination of ROS levels
We first detected the level of ROS by microscopic fluorescence imaging. In brief, the cell culture slides were placed in the six-well plate before the cells were cultured. After siRNA treatments, the culture medium was removed. Subsequently, 1 ml DMEM/F12 culture medium containing 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime) was added to each well of the six-well plate. After incubation for 30 min in a 38.5 • C incubator, the culture slides were washed three times with PBS and mounted onto glass slides. Finally, the fluorescence intensity of the cells was observed by a fluorescence microscope (Olympus). The relative fluorescence intensity level was measured as the average fluorescence intensity of individual cells from three independent experiments in three randomly selected fields.
Meanwhile, the changes in the ROS level were also detected with a ROS Assay Kit (Beyotime) according to the manuals by the flow cytometry as we described ).

Immunofluorescence detection
The cell culture slides were placed into the six-well plate before the cells were cultured. After siRNA treatment, the culture medium was

Cell expansion detection
COCs were cultured in TCM199 with 20 mM bicarbonate containing 0.2 mM sodium pyruvate, 0.5 μg/ml follicle-stimulating hormone, 5.0 μg/ml luteinizing hormone, 10 μg/ml gentamicin, 10% FBS supplemented with si-NC and si-TBX2 and covered with mineral oil at 38.5 • C in a humidified atmosphere of 5% CO 2 in the air. Cell expansion was detected at 0 and 24 h through optical microscopy (Motic). Image-Pro Plus software was used to calculate the average area of the expansion.

Cholesterol and lactic acid detection
Briefly, the cells were cultured into the six-well plate. After siRNA treatment, the culture medium was removed, and the cells were washed twice with PBS. Cholesterol and lactic acid were detected according to the instructions from total cholesterol assay kit and lactate dehydrogenase activity quantitative assay kit (Applygen). Finally, Multi-scan Spectrum was used to detect the optical density value.

Western blotting analysis
The cells were cultured and treated with si-TBX2 or si-NC in six-well plates as described above. After removing the culture medium, cell lysis buffer containing protease inhibitors (Beyotime) was added to the well, and the samples were ultrasonicated for 60 s (three times per second) with an ultrasound transducer. After sonication, the cell sample mixtures were harvested and were placed on ice for 30 min.
Then, the mixtures were centrifuged for 60 min at 13,000 × g, and the

Adenosine triphosphate (ATP)-level detection
The ATP level was determined by an ATP assay kit (Beyotime) according to the manufacturer's protocol. Briefly, after the culture of the siRNA treatment, the cells were collected and lysed with lysis buffer.
Then, the cell lysates were centrifuged at 12,000 × g at 4 • C for 10 min.
After that, the supernatant was obtained. Next, 100 μl of ATP working solution and 20 μl of supernatant were mixed well and added to a 96well plate (Nest Biotechnology). At last, the luminance of the mixture was quantified by an Infinite M200 microplate reader (Tecan).

Statistical analysis
The results were obtained from three repeated independent experiments and were expressed as means ± standard deviation. Data obtained from two groups were compared using the Student's t test.
All statistical analyses were performed using SPSS version 22.0 (IBM) software. P < 0.05 and P < 0.01 were considered to indicate significant differences.

Inhibition of TBX2 reduces cell proliferation and disrupts the cell cycle in bovine cumulus cells
After comparison, the most effective and stable siRNA (siRNA-708) was selected for subsequent experiments (Supplementary Figure S1). Figure 1a, the cell cycle was significantly changed after TBX2 inhibition, and the proportion of G1 phase cells in the TBX2inhibited group increased to 1.38 ± 0.05 times that in the control group (P < 0.01). The proportion of S phase cells decreased to 0.51 ± 0.04 times that in the control group (P < 0.01). The proportion of G2 cells increased to 1.76 ± 0.07 times that in the control group (P < 0.01). The expression levels of cyclin-dependent kinase (CDK)1 and CDK4 genes increased to 1.77 ± 0.20 (P < 0.01) and 2.09 ± 0.23 times (P < 0.01) than in the control group, respectively. The CDK6 gene was downregulated 0.64 ± 0.11 times (P < 0.01), while the expression of the CDK2 gene had no significant change (P > 0.05, Figure 1b). In addition, the cell index in the TBX2-inhibited group was lower than that of the control group after approximately 5 h, and this effect lasted for at least 40 h ( Figure 1c). After TBX2 inhibition, the apoptosis rate of cumulus cells increased from 12.48 ± 2.50% to 19.61 ± 1.95% (Figure 1d). Compared with the control group, the BCL2-associated X (BAX)/B-cell lymphoma 2 (BCL2) level in the TBX2 inhibition group increased by 1.53 ± 0.11 times (Figure 1e).

TBX2 inhibition leads to an increase in ROS accumulation in bovine cumulus cells
ROS are important factors that induce apoptosis in cells. Therefore, we tested whether TBX2 regulates apoptosis by affecting intracellular ROS accumulation. As shown in Figures 2a and 2b, the DCFH fluorescence levels in the TBX2 inhibition group were 1.37 ± 0.09-fold higher than those in the si-NC group. Furthermore, flow cytometry analysis ( Figure 2c) indicated that the intracellular ROS levels of the TBX2 inhibition group were significantly increased to 1.46 ± 0.12 times (P < 0.01) those of the si-NC group. These suggested that inhibition of TBX2 caused ROS accumulation in bovine cumulus cells.

Inhibition of TBX2 disrupts mitochondrial function
As shown in Figure 3a,b, the ΔΨm of the average cell was calculated as a ratio of red fluorescence intensity (J-aggregates; corresponding to activated mitochondria) to green fluorescence intensity (J-monomers; corresponding to inactive mitochondria). The results showed that after inhibition of TBX2, the ΔΨm decreased to 0.38 ± 0.05 times, compared to those in the si-NC group (P < 0.01). In addition, the ATP level

Inhibition of TBX2 increases autophagy levels
Autophagy, which is usually measured by the levels of LC3B, maintains microenvironment stability in vivo, thereby reducing damage to the cells. After inhibition of TBX2, the immunofluorescence results showed a significant increase in the number of cytoplasmic LC3B dot ( Figure 4a). The western blot results were also consistent with this finding. The relative level of LC3BII/I in the TBX2-inhibited group was 1.71 ± 0.26-fold higher than those in the si-NC group (P < 0.05, Figure 4b).

TBX2 inhibition prevents cumulus cell expansion
The expansion level of cumulus cells is an important marker of oocyte maturation. As shown in Figure 5a,b, the relative expansion level of cumulus cells in the TBX2-inhibited group was 0.80 ± 0.31-fold lower than si-NC group (P < 0.05) when oocytes matured. Meanwhile, the mRNA expression levels of the cumulus cell expansion-related genes prostaglandin-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3), and hyaluronan synthase 2 (HAS2) were also significantly decreased by 0.63 ± 0.11, 0.43 ± 0.08, and 0.72 ± 0.14 times, respectively, in the si-TBX2 group, compared with the si-NC group (P < 0.01, Figure 5c). In addition, relative cholesterol levels were reduced by 0.58 ± 0.02-fold (P < 0.01), and relative lactic acid levels were reduced by 0.68 ± 0.03fold in the TBX2 inhibition group compared with the si-NC group (P < 0.01. Figure 5d,e).

DISCUSSION
In this study, we inhibited the expression of the TBX2 gene to explore the physiological role of TBX2 in bovine cumulus cells. In general, after TBX2 was inhibited, the cell cycle was disrupted, the intracellular oxidative stress and autophagy levels were increased and the rate of cell apoptosis was also increased, suggesting that TBX2 can regulate the physiological functions of bovine cumulus cells.
The cell cycle plays an important role in cell proliferation and apoptosis (Pietenpol & Stewart, 2002;Ye et al., 2017). Previous studies had shown that inhibition of TBX2 resulted in an increase in G1 phase cells and a decrease in S phase cells (Pan et al., 2015;Yi et al., 2017b). In this study, changes in cyclin-dependent kinase 2 (CDK2) expression may regulate the G1/S phase transition, as well as DNA synthesis and replication in S phase (Ferguson & Maller, 2010;Ohtsubo et al., 1995). However, the expression of CDK2 did not change significantly. This may be because TBX2 promoted cell cycle progression through cyclin D1 and retinoblastoma protein-E2 transcription factor 1 but not p21 and CDK2 (Pan et al., 2015).
The differential expression of CDK1 indicates that inhibition of TBX2 can promote entry into M phase and the transition from G2 to M phase, thus contributing to mitotic progression in cell division (Barr & Gergely, 2007;Ito, 2000). In addition, inhibition of TBX2 may also be associated with D-type cyclins (D1, D2, and D3) and CDK4/6 and disrupt the essential processes for entry into G1 phase (Sherr & Roberts, 2004). In the G1 phase, CDK4/6-cyclin D promotes cell cycle progression by means of retinoblastoma protein phosphorylation and sequestration of p21 and p27. This indicated that inhibition of TBX2 reduced the release of CDK2-cyclin E complexes as well as CDK2 kinase activity (Bai et al., 2017). In addition, these results are also consistent with existing reports and our subsequent findings that inhibition of TBX2 affects cell proliferation.
Changes in cell proliferative capacity are influenced by nutrient utilisation, mitochondrial function, and the physiological state of cells, which ultimately affect the biological functions of cells (Mandal et al., 2011). Combined with the results of previous studies, our findings suggest that TBX2 may have a potential regulatory effect on the physiological and secretory functions of cumulus cells (Fu, Chen, Li, et al., 2019).
Apoptosis can also be initiated by decreased mitochondrial activity and ROS-induced oxidative stress in addition to abnormal changes in the cell cycle (Atsumi et al., 2006). In this study, inhibition of TBX2 significantly increased BAX/BCL2 levels and the apoptosis rate. This is in accordance with the findings of another study in which TBX2 overexpression reduced caspase 3 cleavage and induced BCL2 upregulation (Yi et al., 2017a). In addition, ROS accumulation is associated with mitochondrial fission and affects cell proliferation and apoptosis (Cadenas, 2018;Wang et al., 2017;Zorov et al., 2014). Given the MMP and ROS assay results, we suspect that inhibition of TBX2 disrupts the mitochondrial fission/fusion balance in cumulus cells (Yi et al., 2017b), which results in an increase in intracellular ROS levels and a decrease in mitochondrial function. Subsequently, the level of autophagy can be upregulated by cells to adapt to the adverse internal environment (Twig et al., 2008), which indicates that the level of TBX2 is important for maintaining environmental stability and generating the building blocks necessary for macromolecular synthesis, energy production, and cell survival (Lee et al., 2014).
Finally, we examined the potential effects of TBX2 inhibition on the expansion and physiological function of cumulus cells. The expansion of cumulus cells is closely related to the development and maturation of oocytes (Su et al., 2009). In this study, inhibition of TBX2 reduced cumulus cell expansion, consistent with the observed downregulation of important genes affecting cumulus cell expansion, such as PTX3, PTGS2, and HAS2 (Fang et al., 2019;Kahraman et al., 2018). Previous studies showed that oocytes are unable to synthesise cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway (Payne & Hales, 2004;Su et al., 2008). The expansion of cumulus cells has a substantial regulatory effect on oocyte secretion, which is associated with metabolic processes in COCs, especially cholesterogenesis (Su et al., 2009;Sugiura et al., 2007). In this study, inhibition of TBX2 led to a decrease in the cholesterol level, which indicates that inhibition of TBX2 will lead to a decrease in precursor substances for the synthesis of steroid hormones (Miller, 2017). This also suggests that TBX2 may have a regulatory effect on the secretory function of cumulus cells and affect oocyte maturation and fertilisation (Bunel et al., 2014;Watanabe et al., 2013). Therefore, we hypothesised that TBX2 would potentially regulate the cumulus cell contact and secretory function that will have a definite impact on the cumulus-oocyte microenvironment (Diogenes et al., 2017), thereby playing a potential role in oocyte development, maturation, and subsequent fertilisation.

CONCLUSION
In conclusion, the results of this study showed that TBX2 plays important roles in regulating cell proliferation, expansion, apoptosis and autophagy and maintaining the mitochondrial function and cholesterol generation of bovine cumulus cells. The relative level of lactic acid was decreased in TBX2-inhibited groups. Significant differences are represented with * (P < 0.05) and ** (P < 0.01).